The research project concerns the mechanism of biosynthesis of vitamin B1 (thiamine) in bacteria. Specifically, the pathway between aminoimidazole ribotide (AIR) and 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP), a portion of thiamine, is under investigation. The approach to be used is to obtain various mutants of Salmonella typhimurium blocked in the conversion of AIR to purines and also blocked between ARI and MAHMP. This should allow for the accumulation of intermediates along the latter pathway. AIR labeled with carbon-14 on carbon-13 will be administered to the bacteria to facilitate identification and characterization of the such intermediates. Also under study will be the compounds that are the precursors of the methyl group and the extra 2-carbon unit included in MAHMP, i.e. carbon-5 and its hydroxymethyl group. Experiments using a pure mutant that excretes MAHMP will be performed and possible precursors will be labeled with carbon-13 or carbon-14. MAHMP obtained will be then analyzed to determine the site of any incorporations.